The high affinity receptor for IgE (E-receptor) on mast cells and basophils plays a central role in immediate hypersensitivity reactions. Reaction of receptor-bound IgE with polyvalent antigen clusters the receptors and this stimulates cellular secretion of preformed and newly synthesized mediators of inflammation. We study the molecular mechanisms by which E-receptors generate such responses. During the past year, our principal studies have been along the following lines: 1) We continued to analyze the interaction of Lyn kinase with E- receptors permanently transfected into Chinese Hamster Ovary cells. A quantitative study now published shows that as little as a single molecule of Lyn kinase per aggregate is sufficint to promote phosphorylation of the receptos, which serves as the initial event. We also initiated experiments employing chimeric proteins consisting of the ecto- and transmembrane domains of an irrelevant receptor chain (IL-2 alpha chain) fused to portions of the E-receptor or of Lyn kinase transfections to clarify the E-receptors interaction with the kinase. Initial results indicating that we could achieve variable levels of expression and evidence for a specific interaction were confirmed and extended. The interactions we observed did not appear to require localization to specialialized domains of the plasma membrane; 2) The transfection studies revealed that as the ratio of the kinase:receptor approaches the level seen with the standard mast cell line (RBL-2H3) we have regularly used in the past, spontaneous phosphorylation of the receptor in the transfectants (but not in the RBL cells) is observed. We are exploring the mechanism for this unexpected difference. These quantitative studies also allowed us to determine that a single molecule of the Lyn kinase per aggregate, can initiate signaling. 3) Earlier studies showed that signaling by the receptor was subject to kinetic proofreading. In such a regimen the signaling is controlled by the lifetime of the initial ligand:receptor interaction. We have now found that under conditions favoring the persistence of signaling (see (4)), even ligands that interact relatively weakly can stimulate late events such as degranulation and synthesis of mRNA coding for various mediators. We are exploring the basis for this apparent paradox. In a second study under this topic, we are examining whether the sequential phosphorylation of tyrosines on a critical early component differs for the high affinity and low affinity ligands. 4) Stimuli generating large-scale clustering of the receptors are short-lived. Our new studies suggest that entanglement of the aggregated receptor with cytoskeletal structures leads to such rapid down-regulation of activated receptors. The basis for this spontaneous inactivation is under investigation. 5) The initial analysis of the changes in the profile of genes that are expressed in activated versus resting mast cells using Serial Analysis of Gene Expression (SAGE) methodology was published. Identifying the function played by some of the newly activated genes has been frustrating, but several leads are being pursued. The similarity of E-receptors to other receptors of the immune system (e.g. the clonotypic receptors on T and B lymphocytes), make it likely that the significance of our studies extends beyond the IgE/mast cell system. - signal transduction, Fc receptors, RBL cells, src-family kinase, protein tyrosine phosphatases, aggregation, ligand antagonists